Tumor necrosis factor receptor associated factor 6 (TRAF6) is central to host immune responses and is important for protective responses to pathogens. We report here a non-canonical role for TRAF6. The tick- borne flaviviruses (TBFVs) have evolved a unique mechanism to use TRAF6 to benefit infection. Understanding how the balance between TRAF6?s antiviral and proviral effects is tipped in favor of TBFV replication may reveal how flaviviruses overcome the cellular antiviral environment, to ultimately cause disease. We identified an interaction between TRAF6 and the TBFV protease, NS3pro. TRAF6 is an E3 ubiquitin ligase that is normally associated with antiviral functions in the context of virus infections. Surprisingly, TBFVs use TRAF6 to replicate in cells. Here, we show that TBFV NS3pro protein interaction with TRAF6 during infection supports TBFV replication. The proviral role of TRAF6 was not seen with mosquito-borne flaviviruses, consistent with the lack of a conserved TRAF6 binding motif. Disruption of the TRAF6-NS3pro interaction by site-directed mutagenesis resulted in a significant reduction of mature protease protein expression, and attenuation of in vitro virus replication. To investigate the TRAF6 proviral function, we assessed the ubiquitination status and found that NS3pro is ubiquitinated in the presence of TRAF6. The overall objective of this application is to determine how NS3-TRAF6 interaction affects protease structure and function(s), virus replication, and pathogenesis in a mouse model of TBFV infection. We hypothesize that NS3pro is activated by TRAF6 through direct interaction and ubiquitination, ultimately to promote productive virus replication. To test our hypothesis, we will use two specific aims. 1) To determine how TRAF6 benefits TBFV replication we will use cell biology and biochemical approaches to determine how NS3pro is modified by ubiquitination and how ubiquitination affects the structure and function of NS3pro. 2) To determine the proviral role of TRAF6 in primary cells (e.g., dendritic cells and neurons) and in pathogenesis in vivo we will use therapeutic and genetic methods to manipulate TRAF6 expression in primary cells and in a mouse model of TBFV pathogenesis. By understanding how TRAF6 benefits NS3pro and its role during infection, we will gain insight into the regulation of a critically important viral enzyme. Then by using a mouse model of TBFV infection we will determine the extent to which the NS3-TRAF6 interaction may be targeted for flavivirus drug development.